Overexpression, purification and biochemical studies of Sortase A from Enterococcus faecalis (Ef) and its inhibition...

Sortase A (SrtA) is a bacterial transpeptidase that garnishes the bacterial surface by adding various virulent factors or proteins by cleaving the LPXTG-specific motif between T and G amino acids. These virulence factors assist in the attachment of host cells, which are essential for bacterial virulence. Enterococcus species are among the multidrug-resistant bacteria that cause nosocomial infections; they have drawn a lot of attention recently. SrtA from E. faecalis (Ef) plays a critical role in pathogenesis, making it a suitable target for the development of antibacterial agents. Since SrtA is not involved in bacterial growth and is present on the surface of bacteria, the probability of developing antibiotic resistance is minimal. In this work, we have done cloning, expression and purification of Ef-SrtA using IMAC (Immobilised Metal Affinity Chromatography) followed by Gel filtration chromatography. Purified Ef-SrtA showed maximum activity at pH-8 and temperature between 45 and 55 °C. The fluorescent assay for kinetic studies of Ef-SrtA showed V_max 3.852 µM.min^-1 and k_cat 7.7 × 10^-2s^-1 for the hydrolysis of substrate using Abz-LPETG-K(Dnp)-NH₂. We have selected fifteen Aloe vera extracted compounds and performed virtual screening and docking experiments to identify potential inhibitors against Ef-SrtA. Among fifteen molecules, Aloenin-a which was bound to the active site with a binding energy of -6.1 kcal/mol, interacted with the active site residues, Arg139, Pro105, Leu39, Ala46, and Cys126. Aloenin-a showed a significant inhibitory effect against Ef-SrtA, with an IC₅₀ value of 20.68 µM. Aloenin-a inhibits biofilm formation at concentrations of 20-250 µg/mL. The fibrinogen assay showed adherence to fibrinogen was reduced in the presence of Aloenin-a for E. faecalis. The results demonstrated that Aloe vera extracts containing Aloenin-a can be a significant antagonist of Ef-SrtA.